updates on prion protein purification donovan duggan bow suriyamongkol supervisor: dr. david wishart...
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Updates on Prion Protein Purification
Donovan DugganBow Suriyamongkol
Supervisor: Dr. David Wishart
Prion Group Meeting2 February, 2007
Donor
Purification continues
Optimisation of purification
Steps to remove contaminating upper and lower bands
•Gradient Elution
•Possible double his tag, combined with a gradient elution
Post purification protocols
1) Determine protein concentration
2) Determine optimal buffer for highly concentrated samples
3) Determine protein structure during each step of post purification protocols
1) Determine protein concentration
• All quantification methods are rough approximations.
Absorbance at 280
Hartree-Lowry and modified Lowry Protein assays.
Bicinchoninic Acid (BCA)
Bradford
Kjeldahl
Very rough approximations with visualization in gels or on filter papers.
2) Determination of optimal buffers
• Heavy empirical screen.
Temperature
pH
Ionic strength
Detergents
Glycerol content
Small stabilizing molecules
3) Determine protein configuration before and after any condition
change.
• Circular Dichroism
• Microscopy
W/T ( 2 mg/mL) Day 1 Day 2 Day 1 Day 2
4C 37C 4C 37C
ddH2O O O Hydrophobic reaction
25 mM NH4HCO3 pH 8.0 O O (20mM Tris, 100mM
50 mM NH4HCO3 pH 8.0 O O NaCl, pH 8.0 base buffer)
100 mM NH4HCO3 pH 8.0 O O Glycerol 5% (v/v) O O
Glycerol 10% (v/v) O O
Disulfide bridges Glycerol 15% (v/v) O O
(20mM Tris, 100mM Glycerol 20% (v/v) O O
NaCl, pH 8.0 base buffer)
DTT 5mM O O Ionic reaction
DTT 10mM O O (20mM Tris, pH 8.0
DTT 15mM O O base buffer)
DTT 20mM O O NaCl 100 mM X
NaCl 200 mM O O
NaCl 500 mM O O
KCl 100mM O O
KCl 200mM O O
KCl 500mM O O
Na2PO4 100mM O O
Na2PO4 200mM O O
Na2PO4 500mM N/A N/A
Wild Type (3 mg/mL)
198 ( 4 mg/mL) Day1 198 (2 mg/mL) Day1 Day2
4C 4C 3h RT
Disulfide bridges Disulfide bridges
(20mM Tris, 100mM (20mM Tris, 100mM
NaCl, pH 8.0 base buffer) NaCl, pH 8.0 base buffer)
DTT 5mM X DTT 5mM O O
DTT 10mM X DTT 10mM O O
DTT 15mM X DTT 15mM O O
DTT 20mM X DTT 20mM O O
Hydrophobic reaction Hydrophobic reaction
(20mM Tris, 100mM (20mM Tris, 100mM
NaCl, pH 8.0 base buffer) NaCl, pH 8.0 base buffer)
Glycerol 5% (v/v) X Glycerol 5% (v/v) O O
Glycerol 10% (v/v) X Glycerol 10% (v/v) O O
Glycerol 15% (v/v) X Glycerol 15% (v/v) O O
Glycerol 20% (v/v) X Glycerol 20% (v/v) O O
Ionic reaction Ionic reaction
(20mM Tris, pH 8.0 (20mM Tris, pH 8.0
base buffer) base buffer)
NaCl 100 mM X NaCl 100 mM O O
NaCl 200 mM X NaCl 200 mM O O
NaCl 500 mM X NaCl 500 mM O O
KCl 100mM X KCl 100mM O O
KCl 200mM X KCl 200mM O O
KCl 500mM X KCl 500mM O O
Na2PO4 100mM X Na2PO4 100mM O O
Na2PO4 200mM X Na2PO4 200mM O O
Na2PO4 500mM X Na2PO4 500mM O O
F198S (4 mg/mL and 2 mg/mL)
178 ( 4 mg/mL) Day 1 Day 2 Day 3
4C RT 37C
10 mM K2PO4
pH 6.0 X
pH 6.6 X
pH 7.2 X
50 mM K2PO4
pH 6.0 X
pH 6.6 X
pH 7.2 X
100 mM K2PO4
pH 6.0 X
pH 6.6 X
pH 7.2 X
10 mM NaOAc
pH 4.0 O O O
pH 4.5 O O O
pH 5.0 O O O
pH 5.5 X
25 mM NaOAc
pH 4.0 O O O
pH 4.5 O O O
pH 5.0 O O O
pH 5.5 X
50 mM NaOAc
pH 4.0 O O O
pH 4.5 O O O
pH 5.0 O O O
pH 5.5 X
D178N (4 mg/mL)
200 ( 2 mg/mL) Day 1 Day 2 Day 3
4C RT 37C
10 mM K2PO4
pH 6.0 O O X
pH 6.6 O O X
pH 7.2 O O X
50 mM K2PO4
pH 6.0 O O X
pH 6.6 O O X
pH 7.2 O O X
100 mM K2PO4
pH 6.0 O O X
pH 6.6 O O X
pH 7.2 O O X
10 mM NaOAc
pH 4.0 O O O
pH 4.5 O O O
pH 5.0 O O O
pH 5.5 O O O
25 mM NaOAc
pH 4.0 O O O
pH 4.5 O O O
pH 5.0 O O O
pH 5.5 O O O
50 mM NaOAc
pH 4.0 O O O
pH 4.5 O O O
pH 5.0 O O O
pH 5.5 O O O
E200K (2 mg/mL)
Summary of Solubility of The Proteins
Wild type (2 mg/mL) , pI 8.86 almost all the buffers tested, including ddH2O
F198S (2 mg/mL), pI 8.86 20 mM Tris, 100 mM NaCl, pH 8.0
D178N (4 mg/mL), 9.08 10 to 50 mM NaOAc, pH 4.0-5.0
E200K (2 mg/mL), 9.23 10 to 50 mM NaOAc, pH 4.0-5.5
Donovan’s results for P102L mutant
Sodium acetate buffer (up to 3 days at 37C)- 10 to 100 mM, pH 4.0- 25 to 100 mM, pH 4.5- 50 to 100 mM, pH 5.0- 10 to 50 mM, pH 5.5
P102L ( ? mg/mL) is stable in:
Potassium phosphate buffer (up to 1 day at 37C)- 10 to 100 mM, pH 8.0
SDS-PAGE Gels of Purified Proteins
Wild type
15
20
kDa
Total amount of 45 milligrams per 1L of culture
F198S
1520
kDa
Total amount of 35 milligrams per 1L of culture
Bradford Std Curve
y = 0.8084x + 0.018
R2 = 0.9977
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 0.2 0.4 0.6 0.8 1
BSA (mg/mL)
AB
S 5
95 n
m
Protein Quantification
Acknowledgements
Dr. David WishartValentyna Semenchenko
Thank you.